It is our intention to isolate, characterize, and determine the nucleotide sequence of the region of the Bacillus subtilis genome, which is involved in initiation of chromosome replication. Restriction endonuclease generated fragments of B. subtilis DNA prepared from Nalidixic acid resistant or Novobiocin resistant mutants will be ligated in vitro with that of the E. coli bacteriophage lambda cloning vector lambda Wes-B or with other suitable vectors such as E. coli or B. subtilis plasmids. This cloning procedure will allow us to purify the isolated regions of the B. subtilis chromosome that carry resistance to Nalidixic acid and/or Novobiocin resistance and obtain large quantities of this DNA. We will then use standard biochemical and genetic methods to determine the structural relationship and organization of these genes to the adjacent region of the chromosome which contains the nucleotide sequence(s) at which the origin of chromosomal replication is initiated. The functional assay of a DNA fragment capable of self-replication will be a very powerful tool for these analyses and has the potential of providing a totally new (natural) cloning vehicle for B. subtilis.